Methods for biological substance analysis employing internal magnetic gradients separation and an externally-applied transport force

ABSTRACT

A method of qualitative analysis of microscopic biological specimens is disclosed in which the specimens are rendered magnetically responsive by immunosspecific binding with feuromagnetic colloid. The specimens are placed in a vessel having a feuromagnetic capture structure supported on a transparent wall. The vessel is then placed in a magnetic field for inducing a high gradient region in the vicinity of the capture structure and, in particular, adjacent to the capture structure along the direction of the applied field. The magnetically responsive specimens are collected and immobilized by the high gradient along the wall adjacent to the capture structure for unobstructed microscopic observation. Subpapulations of distinct types of cells can thus be observed and differentiated. As transport force, such as gravity or an externally applied magnetic gradient, may be applied to the vessel to move the specimens into the high gradient region.

CROSS-REFERENCE TO RELATED APPLICATIONS

Priority is claimed herein to U.S. Provisional Application No.60/019,282, filed Jun. 7, 1996, and to U.S. Provisional Application No.60/030,436, filed Nov. 5, 1996, both of which are incorporated byreference herein.

FIELD OF THE INVENTION

The present invention relates to methods for separating, immobilizing,and analyzing magnetically responsive biological substances from withina fluid medium. More particularly, the invention relates to analysismethods employing a high internal gradient magnetic capture structureformed within a vessel, in conjunction with an externally-applied forcefor transporting magnetically responsive material toward the capturestructure.

BACKGROUND OF THE INVENTION

A magnetic material or magnetic dipole will move in a magnetic fieldgradient in the direction of increasing highest magnetic field strength.Magnetic gradients employed in fluid separations are broadly dividedinto two categories. Internal magnetic gradients are formed by inducinga magnetization in a susceptible material placed in the interior of aseparation vessel. External gradients are formed by an externallypositioned magnetic circuit.

In the case of a simple rectangular bar magnet, field lines which formmagnetic circuits conventionally move from North to South and are easilyvisualized with iron filings. From this familiar experiment inelementary physics it will be recalled that there is greater intensityof field lines nearest the poles. At the poles, the edges formed withthe sides and faces of the bar will display an even greater density orgradient. Thus, a steel ball placed near a bar magnet is first attractedto the nearest pole and next moves to the region of highest fieldstrength, typically the closest edge. For magnetic circuits, anyconfiguration which promotes increased or decreased density of fieldlines will generate a gradient. Opposing magnet designs, such as N-S-N-Squadrupole arrangements having opposing North poles and opposing Southpoles, generate radial magnetic gradients.

Internal high gradient magnetic separators have been employed for nearly50 years for removing weakly magnetic materials from slurries such as inthe kaolin industry or for removing nanosized magnetic materials fromsolution. In an internal high gradient magnetic separator, a separationvessel is positioned in a uniform magnetic vessel. A ferromagneticstructure is positioned within the vessel in order to distort themagnetic field and to generate an "internal" gradient in the field.Typically, magnetic grade stainless steel wool is packed in a columnwhich is then placed in a uniform magnetic field which induces gradientson the steel wool as in U.S. Pat. No. 3,676,337 to Kolm. Gradients ashigh as 200 kGauss/cm are easily achieved. The magnitude of the fieldgradient in the vicinity of a wire is inversely related to the wirediameter. The spatial extent of the high gradient region isproportionally related to the diameter of the wire. As will be detailedbelow, collection of magnetic material takes place along the sides ofthe wire, perpendicular to the applied magnet field lines, but not onthe sides tangent to the applied field. In using such a system, materialto be separated is passed through the resulting magnetic "filter". Then,the collected material is washed, and the vessel is moved to a positionoutside the applied field, so that magnetic can be removed, making thecollector ready for reuse.

Table I below indicates the magnitude of a magnetic gradient as afunction of distance R, from the center of a ferromagnetic wire forround wires of different diameters. The gradients are determined byMaxwell's equations, which produce equations I and II for the strengthof the magnetic field about the wires and the gradient of the field. Theequations give the magnitude of these quantities when the wire has aninternal magnetization per unit volume of M. If the wires are composedof "soft" ferromagnetic materials, the magnetization depends on thevalue, B_(ext), of an externally applied field. For any value of M, evenfor a hard ferromagnetic material with constant, uniform magnetization,the dependence on the distance and wire diameter are as shown. Thegradient values listed in Table 1 assume a typical wire magnetizationsuch that 4 π M=10 kiloGauss (kG), a value close to that of a rare earthmagnetic alloy. Table 1 demonstrates that for a narrower wire, the fieldgradient at the surface of the wire is larger than for thicker wires,although the magnitude of the gradient falls off much more rapidly withdistance from the wire.

                  TABLE I                                                         ______________________________________                                        Diameter of wire                                                                       0.2 μm                                                                              2.0 μm                                                                             20 μm                                                                             200 μm                                                                           2000 μm                             Distance from                                                                                grad B                                                                                grad B                                                                                       grad B                                                                               grad B                           wire center (R)                                                                            (kG/cm)                                                                               (kG/cm)                                                                                   (kG/cm)                                                                           (kG/cm)                                                                              (kG/cm)                           ______________________________________                                        0.1 μm                                                                              600,000  --      --     --    --                                     0.2 μm                                                                                           --00                                                                                           --                                                                                      --                           0.5 μm                                                                                           --00                                                                                           --                                                                                     --                            1.0 μm                                                                                              60,000                                                                               --                                                                                   --                                                                                     --                            2.0 μm                                                                                               7,500                                                                                --                                                                                  --                                                                                 --                                5.0 μm                                                                                              480                                                                                  --                                                                                          --                              10.0 μm                                                                                             60                                                                                    6,000                                                                               --                                                                                      --                           20.0 μm                                                                                           7.55                                                                                   750                                                                                            --                           50.0 μm                                                                                          0.4848                                                                                 48                                                                                           --                              0.l0 mm               0.0606                                                                                 6.0                                                                                            --                            0.20 mm             0.0075075                                                                              0.75                                                                                              --                           0.50 mm           0.00048000048                                                                           0.048                                                                                    4.8                                                                                    --                            1.0 mm                  0.00006                                                                           0.006                                                                                    0.6                                                                                    60                            2.0 mm                  ...                                                                                  0.00075                                                                             0.075                                                                                  7.5                             5.0 mm                  ...                                                                                  0.000048                                                                           0.0048                                                                                 0.48                             ______________________________________                                    

    B.sub.int =[B.sub.ext (μ-1)D.sup.2 ]/[4(μ+1)R.sup.2 ]=2 π M D.sup.2 /4R.sup.2                                         (I)

    grad B.sub.int =[B.sub.ext (μ-1)D.sup.2 ]/[4(μ+1)R.sup.3 ]=2 π M D.sup.2 /4R.sup.3                                         (II)

where D=the diameter of a circular wire

R=the distance from the center of the wire

M=the wire magnetization

μ=the magnetic permeability of the wire

B_(ext) =the magnitude of the external field perpendicular to the wire

B_(int) =the magnitude of the resultant internal field contribution

grad B_(int) =the magnitude of the resultant internal field gradient

A method and apparatus for separating cells and other fragile particlesare described by Graham, et al in U.S. Pat. No. 4,664,796. The apparatuscontains a rectangular chamber within a cylinder. One pair of opposingsides of the chamber are made of non-magnetic material, while the othersides are made of magnetic material. The flow chamber is packed with amagnetically responsive interstitial separation matrix of steel wool.The material to be separated is run through the chamber, which ispositioned in a uniform magnetic field. During separation, the chamberis aligned in the magnetic field such that the magnetic sides of thechamber are parallel to the applied field lines, thus inducing highgradients about the interstitial matrix in the chamber. When the chamberis in this position, magnetically labeled cells are attracted to thematrix and held thereon, while the non-magnetic components are eluted.The chamber is then rotated, so that the magnetic sides face magnets,which "shunts" or "short-circuits" the magnetic field, reclines thegradients in the flow chamber, and allows the particles of interest tobe removed by the shearing force of the fluid flow.

Other internal magnetic separation devices are known. Commonly ownedU.S. Pat. No. 5,200,084 teaches the use of thin ferromagnetic wires tocollect magnetically labeled cells from solution. U.S. Pat. No.5,411,863 to Miltenyi teaches the use of coated steel wool, or othermagnetically susceptible material to separate cells. U.S. patentapplication Ser. No. 08/424,271 now abandoned by Liberti and Wangteaches an internal HGMS device useful for immobilization, observation,and performance of sequential reactions on cells.

External gradient magnetic separators are also known for collectingmagnetically responsive particles. External devices are so-named becausein such devices, a high gradient magnetic field is produced by asuitable configuration of magnets positioned external to the separationvessel, rather than by an internal magnetic structure. A standard barmagnet, for example, produces a gradient because the magnetic fieldlines follow non-linear paths and "fan out" or bulge along respectivepaths from North to South. Typical gradients of about 0.1 to 1.5kGauss/cm are produced by high quality laboratory magnets. Theserelatively low gradients can be increased by configuring a magneticcircuit to compress or expand the field line density. For example, asecond bar magnet positioned in opposition to a first magnet causesrepulsion between the two magnets. The number of field lines remains thesame, but they become compressed as the two magnets are moved closertogether. Thus, an increased gradient results. Adding magnets ofopposing field to this dipole configuration to form a quadrupole furtherincreases the extent of the high gradient region. Other configurations,such as adjacent magnets of opposing fields, can be employed to creategradients higher than those caused by a bar magnet of equivalentstrength. Another method of increasing gradients in external fielddevices is to vary the shapes of the pole faces or pole pieces. Forexample, a magnet having a pointed face causes an increased gradientrelative to a magnet having a flat pole face.

U.S. Pat. No. 3,326,374 to Jones and U.S. Pat. No. 3,608,718 to Aubreydescribe typical external gradient separators. Dipole configuredseparators for preventing scale and lime build up in water systems aredescribed in U.S. Pat. No. 3,228,878 to Moody and U.S. Pat. No.4,946,590 to Herzog. Adjacent magnets of opposing polarity have beenused in drum or rotor separators for the separation of ferrous andnon-ferrous scrap, as described in U.S. Pat. No. 4,869,811 to Wolanskiet al. and U.S. Pat. No. 4,069,145 to Sommer et al.

External gradient devices have also been used in the fields of cellseparation and immunoassay. U.S. Pat. Nos. 3,970,518 and 4,018,886 toGiaever describe the use of small magnetic particles to separate cellsusing an actuating coil. Dynal Corp. (Oslo, Norway) produces separatorsemploying simple external magnetic fields to separate carrier beads forvarious types of cell separations. Commonly owned U.S. Pat. Nos.5,466,574 and 5,541,072 disclose the use of external fields to separatecells for solution to form a monolayer of cells or other biologicalcomponents on the wall of a separation vessel. Resuspension and recoveryof the collected material usually requires removal of the collectionvessel from the gradient field and some level of physical agitation.Turning now to the magnetic particles used in such collection devices,superparamagnetic materials have in the last 20 years become thebackbone of magnetic separations technology in a variety of healthcareand bioprocessing applications. Such materials, ranging in size from 25nm to 100 μm, have the property that they are only magnetic when placedin a magnetic field. Once the field is removed, they cease to bemagnetic and can be redispersed into suspension. The basis forsuperparamagnetic behavior is that such materials contain magnetic coressmaller than 20-25 nm in diameter, which is estimated to be less thanthe size of a magnetic domain. A magnetic domain is the smallest volumefor a permanent magnetic dipole to exist. Magnetically responsiveparticles can be formed about one or more such cores. The magneticmaterial of choice is magnetite, although other transition elementoxides and mixtures thereof can be used.

Magnetic particles of the type described above have been used forvarious applications, particularly in health care, e.g. immunoassay,cell separation and molecular biology. Particles ranging from 2 μm to 5μm are available from Dynal. These particles are composed of sphericalpolymeric materials into which magnetic crystallites have beendeposited. These particles because of their magnetite content and size,are readily separated in relatively low external gradients (0.5 to 2kGauss/cm). Another similar class of materials are particlesmanufactured by Rhone Poulenc which typically are produced in the 0.75μm range. Because of their size, they separate more slowly than theDynal beads in equivalent gradients. Another class of material isavailable from Advanced Magnetics. These particles are basicallyclusters of magnetite crystals, about 1 μm in size, which are coatedwith amino polymer silane to which bioreceptors can be coupled. Thesehighly magnetic materials are easily separated in gradients as low as0.5 kGauss/cm. Due to their size, both the Advanced Magnetics and RhonePoulenc materials remain suspended in solution for hours at a time.

There is a class of magnetic material which has been applied tobioseparations which have characteristics which place them in a distinctcategory from those described above. These are nanosized colloids (seeU.S. Pat. Nos. 4,452,773 to Molday; 4,795,698 to Owen, et al; 4,965,007to Yudelson; 5,512,332 to Liberti & Piccoli; 5,597,531 to Liberti, et aland U.S. patent application Ser. No. 08/482,448 to Liberti, et al). Theyare typically composed of single to multi crystal agglomerates ofmagnetite coated with polymeric material which make them aqueouscompatible. Individual crystals range in size from 8 to 15 nm. Thecoatings of these materials have sufficient interaction with solventwater to keep them permanently in a colloidal suspension. Typically,well coated materials below 150 nm will show no evidence of settling foras long as 6 months. These materials have substantially all theproperties of ferrofluids.

Because of the small particle size and strong interaction with solventwater, substantial magnetic gradients are required to separateferrofluids. It had been customary in the literature to use steel woolcolumn arrangements described above which generate 100-200 kGauss/cmgradients. However, it was subsequently observed that such materialsform "chains" (like beads on a string) in magnetic fields, thus allowingseparation in gradient fields as low as 5 or 10 kGauss/cm. Thisobservation led to development of separation devices using large gaugewires which generate relatively low gradients. Large gauge wires can beused to cause ferrofluids to produce uniform layers upon collection. Bycontrolling amounts of ferrofluid in a system, a monolayer can beformed. Magnetically labeled cells can thus be made to form monolayersas described in commonly owned U.S. Pat. Nos. 5,186,827 and 5,466,574.

Analysis of the cellular composition of bodily fluids is used in thediagnosis of a variety of diseases. Microscopic examination of cellssmeared or deposited on slides and stained by Romanowsky or cytochemicalmeans has been the traditional method for cell analysis. Introduction ofimpedance based cell counters in the late 1950s has led to a majoradvance in the accuracy of cell enumeration and cell differentiation.Since then, various other technologies have been introduced for cellenumeration and differentiation such as Fluorescence ActivatedFlowcytometry, Quantitative Buffy Coat Analysis, Volumetric CapillaryCytometry and Laser Scanning Cytometry. Fluorescence based flowcytometryhas improved the ability to discern different cell types inheterogeneous cell mixtures. Simultaneous assessment of multipleparameters of individual cells which pass a measurement orifice at aspeed of up to 1,000 to 10,000 cells/sec is a powerful technology.However, there are limitations of the technology, such as an inabilityto analyze high cell concentration requiring dilution of blood,impracticability of detecting of infrequent or rare cells, and aninability to reexamine cells of interest. To overcome these limitations,clinical samples are typically subjected to various enrichmenttechniques such as erythrocyte lysis, density separation, immunospecificselection or depletion of cell populations prior to analysis byflowcytometry.

Many bioanalytical techniques involve identification and separation oftarget entities such as cells or microbes within a fluid medium such asbodily fluids, culture fluids or samples from the environment. It isalso often desirable to maintain the target entity intact and/or viableupon separation in order to analyze, identify, or characterize thetarget entities. For example, to measure the absolute and relativenumber of cells in a specific subset of leukocytes in blood, a bloodsample is drawn and incubated with a probe, for example a fluorescentlylabeled antibody specific for this subset. The sample is then dilutedwith a lysing buffer, optionally including a fixative solution, and thedilute sample is analyzed by flow cytometry. This procedure for analysiscan be applied to many different antigens. However, the drawbacks tothis procedure become apparent when large samples are required forrelatively rare event analyses. In those situations, the time needed forthe flow cytometer to analyze these samples becomes extremely long,making the analysis no longer feasible due to economic concerns.

One system which attempted to overcome some of the problems with flowcytometers was the so-called "Cytodisk," described in 1985 by DeGrooth,Geerken & Greve (Cytometry, 6: 226-233 (1985)). The authors describe amethod of aligning cells in the grooves of a gramophone disk. The diskwith dried cells was placed on a record player, and the arm of therecord player was outfitted with an optical fiber immediately behind theneedle. The needle kept the optical fiber aligned with the grooves inthe record. The unicellular algae cells (3 microns in diameter) used inthe reported experiment remained in the bottom of the groove, awaitinganalysis by the optical system. The advantages of the Cytodisk includedthat cells could be subjected to multiparameter measurement with nooptical cross-talk, individual cells could be indexed to saidmeasurements, and cells could be measured repeatedly at different levelsof analytical resolution. However, the system required that the cells bedried upon the gramophone record, a non-homogeneous process damaging tomany cells. Even if cells were effectively dried upon the record foranalysis, they would be dead cells. The current invention seeks tocombine some of the benefits provided by the Cytodisk, includingmultiparameter measurement, indexing, and repeated measurement with newfeatures which allow analysis of intact cells, which can be released forculturing or other re-use, including infusion back into a livingorganism.

SUMMARY OF THE INVENTION

This invention relates to the immobilization of microscopic entities,including biological entities, such as cells which enables separation ofsuch entities from a fluid medium, including whole blood, into a definedregion in a collection chamber, such that analysis by automated means ispossible. This invention also provides for the quantitative collectionof magnetically labeled target entities, such that microliter quantitiesof sample can be used to detect target entities, including thoseentities which occur at low frequencies.

In a preferred embodiment of the invention, a collection vessel isprovided in which ferromagnetic lines are supported by adhesion along atransparent wall. The lines have effective diameters of 0.1 μm to 30 μm,resulting in immobilization and alignment of magnetically labeledbiological materials in an ordered array. In a particularly preferredembodiment of the invention, human blood cells are aligned for automatedanalysis.

The methods of the invention employ dual forces for collectingparticles. In one embodiment, target material is brought into range ofthe internal high gradient region by gravity. In another embodiment, asingle applied magnetic field serves dual purposes. The applied fieldcomprises a first, external magnetic gradient which moves magneticallyresponsive particles to a region of a collection vessel. At the sametime, the applied field induces magnetization in a ferromagneticcollection structure, thereby adding a second, internal gradient whichfurther acts upon the magnetically responsive particles to move theminto defined region of the collection vessel for analysis. The vesselmay be oriented such that the external gradient acts in opposition to,or in conjunction with, the influence of gravity upon the targetmaterial.

The methods of the invention have utility in the separation ofbiological entities which include a wide variety of substances ofbiological origins including cells, both eukaryotic (e.g. leukocytes,erythrocytes, platelets, epithelial cells, mesenchymal cells, or fungi)and prokaryotic (e.g. bacteria, protozoa or mycoplasma), viruses, cellcomponents, such as organelles, vesicles, endosomes, lysosomal packagesor nuclei, as well as molecules (e.g. proteins) and macromolecules (e.g.nucleic acids--RNA and DNA). The biological entities of interest may bepresent in at least samples or specimens of varying origins, including,biological fluids such as whole blood, serum, plasma, bone marrow,sputum, urine, cerebrospinal fluid, amniotic fluid or lavage fluids, aswell as tissue homogenates, disaggregated tissue, or cell culturemedium. They may also be present in material not having a clinicalsource, such as sludge, slurries, water (e.g. ground water or streams),food products or other sources. The method of the invention also hasutility in the separation of various bacteria and parasites from fecalmatter, urine, or other sources.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded perspective view of a separation vessel accordingto one embodiment of the invention;

FIG. 2 is an enlarged perspective view of a ferromagnetic grid disposedin the separation vessel of FIG. 1;

FIG. 3 is a side elevational view of an arrangement for viewingcollected cells in the separation vessel of FIG. 1;

FIGS. 4A and 4B are photographs of cells collected in the arrangement ofFIG. 3 under alternate methods of illumination;

FIG. 5A is a perspective view of an alternative embodiment of aseparation vessel containing a ferromagnetic capture structure accordingto the present invention;

FIG. 5B is a sectional view of the separation vessel taken along theline 5B--5B of FIG. 5A;

FIG. 6 is a schematic diagram of the separation vessel of FIG. 5Apositioned in a relatively homogenous horizontal external magneticfield;

FIG. 7 is a computer generated diagram of the effect of the appliedmagnetic field of FIG. 6 upon movement of individual magneticallyresponsive particles disposed throughout the separation vessel.

FIG. 8 is a schematic diagram of the separation vessel positioned in anapplied field having an external gradient;

FIG. 9 is a computer generated diagram of the path component of numerousmagnetically responsive particles in the separator of FIG. 8 due solelyto the external gradient;

FIG. 10 is a computer generated diagram of the paths followed bymagnetically responsive particles in the separation vessel of FIG. 8,taking into account the external gradient, the internal gradient, andgravity;

FIG. 11 is a schematic diagram an arrangement for automated analysis ofcollected target entities within a separation vessel of the type shownin FIG. 5A; and

FIG. 12 is a schematic diagram of a separation vessel for use inperforming sequential reactions and analysis target material capturedtherein.

DETAILED DESCRIPTION OF THE INVENTION

I. General Definitions

Unless otherwise indicated, terms of general usage throughout thepresent specification are defined as follows.

The term "probe", as used herein refers to an antibody or other specificbinding substance which contains or is adapted to include a detectablelabel. Detectable labels include fluorescent, chemiluminescent andradioactive compounds, as well as compounds which have distinct orrecognizable lightscattering or other optical properties. Detectablelabels also include those compounds which are only detectable uponbinding to the characteristic determinant.

The term "ferromagnetic capture structure" as used herein refers to astructure of ferromagnetic material which becomes magnetized in thepresence of a magnetic field to attract magnetically responsiveparticles. The capture structure may be provided in the form of wires,thin strips, lithographically formed strips, or electroplatedferromagnetic material supported on or by a wall of a separation vessel.The ferromagnetic material may include iron, nickel, cobalt, alloys ofthe same, alloys of magnetic rare earth elements, or other paramagneticmaterials. The term "internal gradient" as used herein refers to amagnetic gradient induced by the capture structure when it is placed ina magnetic field. The term "external gradient" refers to a magneticgradient applied solely by a configuration of magnets or pole pieces,external to the separation vessel. Electromagnets can also be used toform magnetic fields useful in the invention.

The term "determinant" is used here in a broad sense to denote thatportion of the biological entity involved in and responsible forselective binding to a specific binding substance, the presence of whichis required for selective binding to occur. The expression"characteristic determinant" is used herein in reference to cells, forexample, to signify an epitope (or group of epitopes) that serve toidentify a particular cell type and distinguish it from other celltypes. Cell-associated determinants include, for example, components ofthe cell membrane, such as membrane-bound proteins or glycoproteins,including cell surface antigens of either host or viral origin,histocompatibility antigens or membrane receptors.

The expression "specific binding substance" as used herein refers to anysubstance that selectively recognizes and interacts with thecharacteristic determinant on a biological entity of interest, tosubstantial exclusion of determinants present on biological entitiesthat are not of interest. Among the specific binding substances whichmay be used in affinity binding separations are antibodies,anti-haptens, lectins, peptides, peptide-nucleic acid conjugates,nucleic acids, Protein A, Protein G, concanavalin A, soybean agglutinin,hormones and growth factors. The term "antibody" as used herein includesimmunoglobulins, monoclonal or polyclonal antibodies, immunoreactiveimmunoglobulin fragments, single chain antibodies, and peptides,oligonucleotides or any combination thereof which specifically recognizedeterminants with specificity similar to traditionally generatedantibodies.

The term "magnetically responsive particles" as used herein refers tomagnetic particles of metallic or organo-metallic composition,optionally coated with polymer, preferably coated with a polymer ofbiological origin such as BSA. The particles may be linked with anantibody or other specific binding substance to allow them to bind tobiological entities of interest. Appropriate magnetic material ismanufactured by Dynal, Rhone Poulenc, Miltenyi Biotec, CardinalAssociates, Bangs Labs, Ferrofluidics, and Immunicon Corp. Also includedin the term "magnetically responsive particles" is a biologicalentity-magnetic particle complex, optionally bound to a fluorescentlabel or other detectable label.

The preferred magnetic particles for use in carrying out this inventionare particles that behave as true colloids. Such particles arecharacterized by their sub-micron particle size, which is generally lessthan about 200 nanometers (nm), and their stability to gravitationalseparation from solution for extended periods of time. Such smallparticles facilitate observation of the target entities via opticalmicroscopy since the particles are significantly smaller than thewavelength range of light. Suitable materials are composed of acrystalline core of superparamagnetic material surrounded by moleculeswhich may be physically absorbed or covalently attached to the magneticcore and which confer stabilizing colloidal properties. The size of thecolloidal particles is sufficiently small that they do not contain acomplete magnetic domain, and their Brownian energy exceeds theirmagnetic moment. As a consequence, North Pole, South Pole alignment andsubsequent mutual attraction/repulsion of these colloidal magneticparticles does not appear to occur even in moderately strong magneticfields, contributing to their solution stability. Accordingly, colloidalmagnetic particles are not readily separable from solution as such evenwith powerful electromagnets, but instead require a magnetic gradient tobe generated within the test medium in which the particles are suspendedin order to achieve separation of the discrete particles. Magneticparticles having the above-described properties can be prepared asdescribed in U.S. Pat. Nos. 4,795,698, 5,512,332 and 5,597,531.

II. Gravitationally-Assisted Internal Gradient Immobilization

Referring now to FIG. 1, there is shown an exploded view of a separationvessel 10 according to a first embodiment of the invention. The vessel10 comprises a pair of opposed parallel wall members 12 and 14 separatedby perpendicular walls 16 defining an interior chamber. A ferromagneticcollection structure comprising a plurality of longitudinally extensivemembers is supported on and by an interior surface of the chamber. Forexample, a nickel mesh 18 is positioned upon the interior surface of thewall 14 and held thereon by an adhesive.

A portion of the mesh 18 is shown in FIG. 2. The mesh is formed byelectroplating techniques, so that there are no interwoven oroverlapping intersections that would undesirably entrap non-targetsubstances by capillary attraction. Suitable meshes include nickel gridsused in electron microscopy and are sold by Polysciences, Inc. ofWarrington, Pa. (for example, catalog #8424N). The mesh comprises aplurality of longitudinal members 18a joined to cross members 18bforming a grid for mechanical support. The separation between thelongitudinal members 18a should be at least twice the diameter of theparticles desired to be collected. When the vessel 10 is positioned in amagnetic field transverse to the longitudinal members 18a,magnetically-labeled target material will be captured along both sidesof each of the longitudinal members 18a. In order to form a monolayeredlinear array of the target particles along the interior surface of thewall 14 supporting the mesh 18, the height of the longitudinal members18a should be no greater than the average diameter of the targetentities. The number of target entities that may be captured in thevessel 10 is equal to twice the total length of the longitudinal members18a divided by the average diameter of the target particles. Theferromagnetic collection structure, and hence the chamber, can thus besized to permit collection of substantially all of the target entitiesexpected to be present in a sample of test fluid.

EXAMPLE 1

Leukocyte Differentiation in Whole Blood

A vessel 10 was constructed having longitudinal members 18a extending150 mm along a region of the wall 14 measuring 5 mm by 3 mm. Thelongitudinal members 18a were 5 μm in height, 20 μm wide, and separatedby 63 μm spaces. The supporting members 18b were 48 μm wide. The heightof the chamber was 0.13 mm, for a chamber volume of 2 μl. Hence, forcollecting leukocytes, which have an average diameter of 10 μm, theparticle collection capacity was 30,000. Such a collection capacity issufficient for collecting substantially all leukocytes in the chambervolume.

A test fluid was prepared by adding 0.4 μg of a 130 nm CD45-labeledferrofluid, 3 ng of acridine orange, and 10 ng of ethidium bromide to 1μl of blood. The test fluid was allowed to incubate for 10 minutes, anddeposited in the vessel 10. The vessel was then placed between a pair ofmagnets 20a and 20b, as shown in FIG. 3, with the mesh 18 positioned atthe bottom of the chamber to allow the labeled cells to settle towardthe mesh under the influence of gravity, and then to be aligned alongthe longitudinal members of the mesh in the internally-generated highgradient regions along the longitudinal members. To improve visibilityof the captured material, the arrangement shown in FIG. 3 can then beinverted to allow the non-target material to settle away from the mesh18.

Acridine orange is absorbed by the nucleated cells, which will emitgreen light when excited by blue light (460-500 nm). Under the sameillumination, intracytoplasmatic granules of granulocytes will emit redlight. Ethidium bromide is absorbed only by cells having non-intactmembranes (i.e. dead cells), and will emit deep red light under blueillumination. The optical response of the material in the chamber toblue illumination was viewed through an inverted microscope 22. Theresulting photographic image of FIG. 4A was obtained under a combinationof blue illumination (permitting visibility of the fluorescence emittedfrom the captured cells) and ambient white illumination (permittingvisibility of the ferromagnetic mesh). The captured fluorescent cellscan easily be distinguished from other blood components.

Discrimination between cell types can be achieved by detection ofselected emission spectra and lightscatter properties of the collectedcell. It will be apparent to those skilled in the art that probes withvarious specificities and different fluorescence excitation and emissioncan be used to differentiate between the captured, aligned cells. Also,one or more excitation wavelengths can be used to discriminate betweenthe targets, or between the targets and the collection structure. Forexample, FIG. 4B shows the same collected cells under monochromatic bluefluorescent light. The collection structure is no longer easily visible,and the collected leukocytes are readily discernible. A standardmicroscope could then be used to observe the cells.

This example illustrates the differentiation of two types of cells. Inthis case, leukocytes were separated from other cell types and among thealigned leukocytes, live cells were discriminated from dead cells by theuse of dyes. It will be apparent to one skilled in the art that any two(or more) cell types can be differentiated using different probes. Forexample: fetal nucleated red blood cells from maternal blood,circulating tumor cells (Epcam⁺ CD45⁻) from normal nucleated bloodcells, platelets (CD41⁺, PAC-1⁻) from activated platelets (CD41⁺,PAC-1⁺), and various leukocyte subsets. Important leukocytes subsetsfound in human blood include CD4⁺ or CD8⁺ cells (T-lymphocyte cells) ;CD56⁺ cells (NK cells); CD19⁺ cells (B-lymphocytes); CD14⁺ cells(monocytes); CD83⁺ cells (dendritic cells) ; CD33⁺, CD66a⁺, or CD64⁺cells (granulocytes); CD66a⁺ CD66b⁺ cells (activated granulocytes);CD34⁺ cells )progenito4r cells); and CD90w⁺ cells (hematopoietic stemcells).

EXAMPLE 2

Immunophenotypic Differentiation in Whole Blood

A blood sample is incubated with a fluorescent nucleic acid dye andferrofluid labeled with an antibody directed against a cell surfaceepitope such as, for instance, CD4 expressed on T-helper lymphocytes andmonocytes or CD34 present on progenitor cells. The incubation can takeplace in or outside the separation vessel. The vessel is then introducedinto a magnetic field and the cells exhibiting the cell surface antigenrecognized by the bioactive ferrofluid align on both sides of theferromagnetic lines. Although all nucleated cells are fluorescentlylabeled, only those which are adjacent to the ferromagnetic lines aretarget cells and will be identified as such by an optical detectionsystem arranged to scan for cells along the lines, as described furtherherein.

When the target cell frequency is low, as is the case for progenitorcells in peripheral blood identified by CD34 in normal donors (1-10CD34+ cells/μl), the likelihood increases that non-target cells presentby coincidence along the ferromagnetic lines will influence the accuracyof the enumeration. The likelihood that non-target cells are present bycoincidence along a ferromagnetic line, and thus mistakenly enumeratedas a target cell, can be reduced by decreasing the total length of theferromagnetic lines. This can be achieved by decreasing the number offerromagnetic lines in the chamber.

An alternative approach is not to use a fluorescent nucleic dye but touse a fluorescent labeled monoclonal antibody or other probes directedagainst the same cell type as the bioactive ferrofluid. Preferably thisprobe is directed against a different epitope as the bioactiveferrofluid. In this approach only the target cells are fluorescentlylabeled and identified as such. A drawback of the latter procedure is,however, that it requires a higher sensitivity of the detection system.Other labelling strategies include those generally used in flowcytometryand referred to as multi-color and/or multidimensional analysis. In thiscase, a bioactive ferrofluid is used to align the particles of interestalong the ferromagnetic lines and a variety of monoclonal antibodies orother antigen specific probes labeled with different fluorochromes areused to identify different characteristics or populations within theimmunomagnetically immobilized particles.

III. External Field-Aided Internal Gradient Immobilization

Separation methods according to a second embodiment of the inventionemploy both internally-generated and externally-applied magneticgradients for collecting and immobilizing magnetically-responsive targetsubstances. A non-uniform magnetic field is applied to a separationvessel. The external magnetic gradient moves magnetically responsiveparticles towards a ferromagnetic capture means. The applied magneticfield also induces magnetization of a ferromagnetic capture structuresupported in the vessel. As the magnetically responsive particles movetowards the ferromagnetic capture structure, they experience theadditional influence of the internally-generated gradient, and are drawntoward the capture structure. If the capture structure is of anappropriate configuration, magnetically responsive particles areimmobilized to align along the capture structure, and can be analyzedthrough a transparent wall of a chamber defined by the separationvessel. If the target material is appropriately labeled, fluorescence orlight scattering can be measured through the wall to quantify the amountof target material in the test sample.

In a preferred embodiment of the invention, the applied magnetic fieldimpels movement of the magnetically responsive particles against theforce of gravity, providing an additional means of separation of labeledfrom unlabeled particles, thus reducing non-specific collection ofparticles.

Target substances labeled with the magnetically responsive colloidalparticles described above can be collected in a collection vessel 10,shown in FIGS. 5A and 5B. The vessel 10 comprises a tub-shaped carriermember 12 having a recess formed therein, and a top wall member 14. Thewall member 14 is configured to fit into the carrier member 12 to definea chamber 11 bounded by the interior surface of the wall member 14 andthe interior surfaces of the carrier member 12. The wall member 14 isformed of a non-magnetic transparent material, such as glass, quartz orclear plastic. The carrier member 12 is also formed of a non-magneticmaterial, and is preferably also transparent.

The wall member 14 covers a portion of the recess formed in carriermember 12 to provide orifices 16a and 16b at opposed longitudinal endsof the chamber 11. The exposed recessed portions 18a and 18b of thecarrier member 12 provide receptacles into which a drop of test fluidmay be placed for analysis. Such a fluid may then flow into the chamber11. Entry of fluid into the chamber 11 can be enhanced by capillaryaction, if the chamber is sufficiently narrow. Fiducial reference marks(not shown) may be formed or imprinted upon the wall member 14 toprovide means for measuring the volume of fluid contained within thechamber 11.

A ferromagnetic collection structure is supported by adhesion or formedupon the interior surface of the wall member 14. In the embodiment shownin FIGS. 5A and 5B, the ferromagnetic collection structure comprises aplurality of lithographically-defined ferromagnetic lines 20 formed uponthe interior surface of the wall member 14. The walls of the chamber 11are optionally coated with a material such as BSA, silicone, or anegatively charged surface coating to provide chemically or biologicallyinert exposed interior surfaces. It is important to eliminate a buildupof electrostatic charge upon the wall surfaces to limit non-specificbinding of target particles or free magnetic material to the walls ofthe chamber.

When the vessel 10 is placed into a magnetic field, the ferromagneticlines 20 will become magnetized. The magnetic gradients produced by suchlines 20 are comparable to the gradients calculated for a circular wirehaving the same cross-sectional area. The width of the ferromagneticlines will not affect the monolayering of the particles along themagnetic lines, but the width does affect the strength of the magneticgradient. The gaps between the lines 20 are preferably at least twicethe diameter of the target particles. Optionally, a single line may beused for collection.

The thickness of the magnetic lines is chosen to be on the order ofmagnitude of the magnetically responsive target entities to becollected, so that the target entities will align along opposite sides 7of the lines in a monolayer. Therefore, the lines 20 may be on the orderof thickness of the particles to be collected. Preferably, the lines 20are thinner than the diameter of the entities to be collected. If theentities to be collected are human lymphocytes on the order of 10 μm indiameter and the ferromagnetic lines 20 are about 5 μm thick, the cellswill align in a single layer about 10 μm thick. It is particularlypreferred for the magnetic lines to be significantly thinner than thediameter of particles to be collected. For example, magnetic lines onthe order of 0.25 μm may be used to collect entities of 10 μm indiameter.

Such thin ferromagnetic lines can be manufactured by methods currentlyused in the manufacture of computer chips. In such a method, the surfaceof the wall member is first coated with a ferromagnetic material by avapor deposition technique, such as vacuum evaporation or sputtering.Such a technique provides a layer of metal adhered to the eventualinterior surface of the vessel. The combination of ferromagneticmaterial and the material used to form the wall member should beselected to provide sufficient adhesion of the ferromagnetic material tothe wall member. A layer of photosensitive polymer, or photoresist, isthen applied to the coated surface of the wall member and exposed to apattern of ultraviolet light corresponding to the desired pattern of theferromagnetic capture structure (or a negative image thereof, dependingupon the photoresist employed). The photoresist is then developed torender undesired portions of the metal coating susceptible to removal byetching, such as wet chemical etching or reactive ion etching.Alternatively, the lines may be formed by a lift-off procedure wherein aphotoresist pattern is first applied to the wall member and is removedsubsequent to deposition of a ferromagnetic coating.

Such lithographic methods may be employed to produce a selected patternof ferromagnetic metallization on a single wall member or upon alarge-area substrate that is later to be divided into a plurality ofwall members. These lithographic techniques can be substantially cheaperthan the use of electroplating or electroforming, which would be usedfor thicker lines. Thin lines, by their nature, also tend to be smootherthan their thicker counterparts. Such consistency is a by-product of themanufacturing technique. Smoother lines are important, because theinduced magnetic fields are likewise more consistent. Since such smalllines are being used, the strength of the magnetic field will varygreatly along a relatively "bumpy" line, which will lead to clumping ofthe collected magnetic material. Thus, a smoother ferromagnetic capturestructure and more consistent magnetic fields will result in more evenlyspaced magnetic material, facilitating automated examination of thecollected material.

The separation of a magnetically responsive target substance, using thevessel 10, shall now be described in connection with various magneticarrangements wherein an exemplary target substance shall be humanlymphocytes labeled with magnetic particles manufactured as described inU.S. Pat. Nos. 4,795,698, 5,512,332 and 5,597,531 and in U.S. patentapplication Ser. No. 08/482,448 now U.S. Pat. No. 5,698,271, issued Dec.16, 1997.

FIG. 6 shows the vessel 10 positioned in a substantially uniformmagnetic field, shown by field lines 30, created in a gap between twomagnets 21 of opposing polarity. For proper magnetization, thelongitudinal axis of the ferromagnetic capture structure is orientedperpendicular to the field lines 30.

FIG. 7 is a computer-generated diagram illustrating the paths 40followed by numerous magnetically responsive particles 41 after thevessel is positioned in a homogeneous magnetic field. Magnetizedferromagnetic lines 20 with a thickness of 5 μm are shown end-on. Amajority of particles in the chamber are unaffected by the internalmagnetic gradients and eventually fall to the bottom of the chamberunder the influence of gravity.

In the computer simulation employed to produce FIG. 7, all of theparticles in the chamber were assumed to be magnetically responsive. Inan actual separation, the majority of cells will not be magneticallyresponsive, and will thus settle to the bottom of the chamber under theinfluence of gravity. The relatively few target cells will collect inrespective linear monolayers along the ferromagnetic lines.

In order to obtain quantitative information about the target particles,a reproducible and high percentage of the particles would desirably becollected by the device. In order to obtain information about relativelyrare events, such as circulating tumor cells, fetal cells in maternalblood, or hematopoietic stem cells, virtually all target particles mustbe collected. In order to use relatively thin ferromagnetic structuresto obtain alignment of the particles, a method of "sweeping up" thecells in the chamber is necessary to move the particles into thespatially limited internal high gradient regions. One way of "sweepingup" particles would be to use a narrow chamber. As indicated in FIG. 7,a chamber thickness of just under 100 μm is sufficient for 5 μmferromagnetic lines, but this would require a small chamber volume,which would limit the opportunity to observe rare species. Using a longchamber to increase volume would require a longer ferromagnetic capturestructure, and would increase the time needed to search along thecapture structure for the collected target material. One couldalternatively turn the chamber upside-down, such that gravity wouldassist to settle all particles upon the wires as described above inconnection with the first embodiment. However, in fluids having aheterogeneous population dominated by non-target species, magneticallylabeled material may not be able to move through a thick layer ofsettled non-target material to reach the ferromagnetic capturestructure, resulting in loss of selectivity and crowding of thedetection area. Another approach would be to increase the field strengthof the magnets, but as shown in formula II, to double the range of thegradient, one would have to increase the external field strengtheight-fold.

One method of the instant invention uses a non-uniform applied magneticfield to magnetize the ferromagnetic capture structure and also toprovide an external gradient perpendicular to the capture structure"sweep up" the magnetically responsive particles not initially locatedwithin the influence of the internal magnetic gradients. The appliedmagnetic field preferably supplies an external gradient of sufficientmagnitude to transport the cells towards the ferromagnetic capturestructure where they are then immobilized against the wall adjacent thecapture structure by the internal magnetic gradient. Attributes of sucha field include that it is substantially homogeneous within a planeparallel to the ferromagnetic capture structure, and that the field isoriented perpendicular to the horizontal longitudinal axis of thestructure. Additionally, the field includes a vertical external gradientcomponent that increases in the direction toward the capture structure,and that the external gradient is high enough to transportmagnetically-labeled material toward the capture structure. A magneticfield which could serve such dual purposes can be produced by variousconfigurations of magnets. One advantageous arrangement of externalsource magnets is shown in FIG. 8.

FIG. 8 shows the separation vessel 10 positioned at a preferred locationrelative to a pair of opposed magnetic poles 23 and 24 having a gapformed therebetween. The lower surfaces of the poles 23 and 24 aretapered toward the gap, so that the magnetic field applied to thechamber is non-uniform, and has a substantially vertical gradienteffective to urge magnetically-responsive particles within the chamberagainst the force of gravity toward the ferromagnetic collectionstructure on the upper wall.

FIG. 9 shows the paths that would be followed by such particles withinthe chamber in the absence of the ferromagnetic collection structure. Ascan be seen, the influence of the externally-applied gradient issufficient to move the particles substantially vertically toward theupper wall of the chamber.

FIG. 10 shows the paths followed by particles within the chamber,including the effect thereon caused by the presence of a ferromagneticcollection structure comprising lithographically-defined lines having athickness of 5.0 μm and a width of 20 μm. As can be seen, the externallyapplied gradient tends to urge particles initially located in the lowerportion of the chamber to move into the high gradient regions generatedby magnetization of the ferromagnetic lines.

The precise design parameters for the magnets 23 and 24 shown in FIG. 8(and shown in connection with an automated observation system in FIG.11) required to induce a desirably strong internal gradient and to applya desirably strong vertical external gradient, will depend uponapplication-specific conditions such as the magnetization of themagnetic particles employed, the mass and size of the target entities,and the viscosity and temperature of the fluid medium. Those skilled inthe art will be enabled hereby to select appropriate design parametersin view of such considerations. In experimental conditions such as aredescribed herein, a pair of rare earth magnets (Crucible Magnets;Elizabethtown, Kentucky) having an internal magnetization of 1200 gaussand an acute taper angle of 20° separated by a distance of 5.0 mm toform a gap through which observations of collected entities could bemade. The upper surface of the separation vessel was positioned 2.0 to3.0 mm below the gap. Although FIG. 10 shows a chamber height of 200 μm,chamber heights of 1 mm have been modeled and actually used to collectand quantify magnetically labeled cells.

Of course, it is possible to exceed the capacity of the magnetic linesby attempting to collect so much target material, that a monolayer oftarget particles is no longer possible. In which case, the dilution ofthe test sample or the use of a larger chamber with a greater linearcapacity of the magnetic lines is required.

Thus, analysis of the magnetically labeled target material is enabled.Alignment of the target material in a monolayer also allows for theanalysis to be conducted by automated means, such as mechanicalautomated cell counting technology. The target material can beilluminated through the transparent wall member and the opticalproperties of the target material can be detected. Optical propertiesinclude the direct observation of the target material and themeasurement of adsorbed, scattered or fluorescent light. Optionally, thetarget material can be analyzed aided by the addition of a substrate orother compound. Other compounds include the use of probes whichrecognize a characteristic determinant of the target material, andnuclear, cytoplasmic or membrane dyes. These probes can be eitherinherently fluorescent, fluorescently tagged, or fluorescent only uponbinding to the characteristic determinant. Differentiation of the targetmaterial, i.e., leukocytes, is thus possible with a specific bindingsubstance which recognizes subsets of the target material.

Although the above descriptions are exemplified by the collection ofleukocytes, it is also possible to immobilize other types of cells inthe apparatus of the invention. For example, platelets can be selectedby use of CD41 or CD61. Differentiation into subsets includes ananalysis of their activation status (recognition by CD62p or PAC-1) orthe presence of granules (recognition by CD63 or LDS-751). Theimmobilization of red blood cells is discussed elsewhere in thisspecification.

A notable advantage of the apparatus and method of the invention is thatthe ability to provide linearly monolayered entities presents theopportunity to perform sequential reactions in a rapid and highlyefficient manner. Thus, not only can the apparatus and method of theinvention be used to facilitate cell analysis for the determination ofcell surface characteristics, e.g., T-Cell, B-Cell Progenitor cells andsubset markers thereof, but they can in principle be applied for theanalysis of intracellular components or genes. Such an analysis would bedone by first capturing and aligning the cells of interest. This stepwould be followed by a series of sequential flow-by reactions, whichwould permeate the cell membrane, tag entities of interest and amplifysignal on tagged entities. This capability provides a distinct advantageover existing cytometric technologies.

The following examples further describe aspects of the presentinvention.

EXAMPLE 3

A direct coated ferrofluid was prepared according to U.S. patentapplication Ser. No. 08/482,448. The ferrofluid particles were coatedwith CD45 antibody, which binds to leukocytes. The ferrofluid was storedin a HEPES buffer, pH 7.5 at 100 μg/ml.

The target cells were CEM cells at a concentration of 5×10⁶ cell/ml. 100μl of cells were incubated with 10-30 μl of ferrofluid for ten minutesat room temperature before loading the collection chamber.

A separation vessel was provided with a chamber having dimensions of 0.1mm×5 mm×20 mm, for a chamber volume of 10 μl. The ferromagnetic capturestructure comprised lithographically formed lines of nickel havingdimensions of approximately 5 μm thick×25 μm wide. The lines were spacedat 100 μm intervals.

After incubation, approximately 10 μl of the magnetically labeled CEMcells were loaded into the collection chamber. The interior of thechamber was first observed in the absence of a magnetic field and almostall of the cells were observed to have settled at the bottom of thechamber. The chamber was then agitated to resuspend the cells. Then, thecollection chamber was placed into a magnetic field formed by two squaremagnets (such as the magnets shows in FIG. 2). The vessel was located ina non-uniform region of the field outside of the substantially uniformfield directly between the magnets (i.e., at the location 33 shown inFIG. 6). Hence, an external gradient in the vertical direction wasapplied to the chamber to urge the magnetically responsive particlestoward the ferromagnetic capture means.

While the collection chamber was positioned in the magnetic field, itwas supported upon a microscope stage for observation of the cells werethrough a transparent wall of the chamber. Almost all of the targetcells were observed to be aligned along the ferromagnetic capture linesin a single layer. Most of the cells were observed to be aligned afterabout ten seconds after the chamber was placed in the magnetic field.After a minute, all discernible cell movement ceased.

In another experiment, the loaded chamber was placed in a substantiallyhomogenous magnetic field as indicated by the chamber 10 shown in FIG.6. Although the magnetized wires collected approximately 50% of themagnetically labeled CEM cells, a large number of cells settled to thebottom of the collection chamber.

EXAMPLE 4

The experiment of Example 3 was repeated with human whole blood. 100 μlof whole blood was incubated with 10-30 μl of the CD45 direct labeledferrofluid described in example 1. After a ten minute incubation at roomtemperature, the collection chamber was loaded with 10 μl of test sampleand positioned in the magnet arrangement described above. A shortsettling period of one minute was required to allow the non-target redcells to settle to the bottom of the chamber and away from theferromagnetic capture means to allow observation of the targetleukocytes cells. Upon microscopic examination, the target cells wereseen to be aligned along upon the ferromagnetic capture lines. In otherexperiments with fluorescently labeled nucleated cells, no settlingperiod was required to distinctly identify the collected target cells(i.e. leukocytes).

Although this example illustrated the alignment of leukocytes in wholeblood, it is possible to further distinguish or differentiate theleukocytes with the addition of probes to the desired subpopulations ofleukocytes. As noted in example 1, it will be apparent to one skilled inthe art that the method described in this example could be applied tonumerous types of cell separation and/or differentiation.

EXAMPLE 5

The instant invention should also be useful for conducting competitiveimmunoassays. Proteins, hormones, or other blood components may bemeasured in whole blood using a device similar to that described inconnection with FIGS. 1A and 1B. Magnetic particles which directly orindirectly bind to the blood components to be analyzed could beintroduced into a blood sample, along with a fluorescent,chemiluminescent or other detectable probe, which binds directly orindirectly with the component to be analyzed. After the solutioncontaining the blood, magnetically responsive particle, and detectableprobe has been introduced into the device, the device is placed into anon-uniform magnetic field, and oriented such that gravity and theexternally applied gradient act together to reinforce each other,instead of acting in opposition. The magnetically labeled protein,hormone, or other blood component will be drawn down to the magneticcollection structure. The excess detectable probe would remain insolution. The cellular components in the blood would also be drawn downtowards the magnetic collection structure due to gravity. Detection ofthe non-bound detectable probe through fluorescence emission,chemiluminescence, or other means would be possible through atransparent wall of the collection device. The probe's signal, such aslight emission, from the non-bound detectable probe would initially beblocked by cells, such red blood cells. Such cells would eventuallysettle to form a layer over the magnetic collection structure(s) toallow unobstructed detection of an emission fluorescence, or lightscattering probe signal.

III. Automated Optical Analysis of Immobilized Target Entities

As noted above in connection with FIG. 6, the fringing field beneath apair of opposed rectangular cross-section magnets is capable ofproviding the desired vertical external gradient while inducing internalmagnetization of the ferromagnetic collection structure. For microscopicobservation of the collected material, wherein the optical observationsystem is limited by a finite focal length such as less than 5 mm, it isdesirable to reduce the vertical distance between the top wall of thecollection vessel and the top of the magnetic elements providing thefield. In general, such an objective can be achieved in a magneticarrangement having two opposing pole faces separated by a gap, whereinthe pole faces are formed to have tapering surfaces toward the gap, suchas shown in FIG. 8.

Providing a desirably short distance between the top of the magneticarrangement and the top of the vessel permits the use of variousautomated observation means. Additionally, because target entities arecollected in an orderly pattern on the interior surface of a transparentchamber, an automated observation system can be configured to providerelative motion between the vessel and the light gathering elements ofthe observation system in order to "track" the collected target entitiesfor automated enumeration, which can include spectral analysis of lightemitted or absorbed by the collected targets.

One such automated analysis system 100 is shown in FIG. 11. The analysissystem 100 comprises optical tracking and beam analysis componentssimilar to those employed for reading compact discs known in the audioand data storage arts. Briefly, a pair of laser diodes 110 and 112 areconnected with a power supply 114 to generate respective parallel beamsof light. One beam is employed by the analysis system for locating andtracking lines of the ferromagnetic collection structure. The other beamis used for detecting the presence of collected target entities adjacentto a located line. Relative motion between the optical elements of theanalysis system 100 is provided by a mechanical tracking unit 116.Coordination of the functions of the analysis system 100 is provided bya microprocessor 118.

Laser 112 generates the tracking beam, which is transmitted throughdichroic mirrors 120 and 122, and focussed upon the upper interiorsurface of the separation vessel 10 by objective lens 124. The trackingbeam is reflected from the interior surface of the separation vessel,and is re-transmitted through dichroic mirrors 122 and 120 toward aphotodetector 126. Photodetector 126 generates an electric signal inresponse to receiving the reflected light, which is provided to themicroprocessor 118. The mechanical tracking unit 116 is operated by themicroprocessor 118 to move the objective in the presumed direction ofthe lines of the ferromagnetic collection structure. Microprocessor 118is programmed to detect deviations within the electrical signal fromphotodetector 126 to provide a feedback signal to the mechanicaltracking unit 116 for adjusting the position of the objective 124 in adirection perpendicular to the lines of the ferromagnetic collectionstructure.

As the objective is moved to track the lines of the ferromagneticcollection structure, laser 110 is operated to generate a beam of lightfor detecting the presence of collected target entities. The light fromlaser 110 is transmitted through dichroic mirrors 128 and 122, and isfocussed upon the upper interior surface of the collection vessel toform a spot adjacent to the focal point of the tracking beam. Lightreflected by target entities will be transmitted through dichroicmirrors 122 and 128 toward photodetector 130. Photodetector 130generates an electrical signal representative of the light reflectedfrom the target entities, which is transmitted to microprocessor 118.Microprocessor 118 is programmed to monitor variations in the electricalsignal from photodetector 130, in order to provide an analysis outputsignal, such as a counting signal at an output terminal 132. It will beappreciated that such an output signal can be further processed toprovide information relative to the quantity and respective positions ofthe collected target entities.

In alternative embodiments, one laser could be used to illuminate thechamber, instead of two lasers as depicted in the analysis system 100.Optionally, the laser could be eliminated entirely and the chamber couldbe illuminated with a light-emitting diode or other light source,including light sources that illuminate the chamber from the sides orfrom below. In other embodiments, spectral analysis components, such asoptical filters and gratings, as well as illuminating components havingvarious spectral characteristics, can be employed in an automatedanalysis system for conducting spectral analysis of light emitted fromthe collected entities as an objective lens of the analysis system ismoved to track the ferromagnetic collection structure.

In some cases, it may be desirable to include means to vibrate thechamber, to prevent magnetic particles from being held by frictionagainst the walls of the chamber. Vibrating the chamber has been foundto increase magnetic separation efficiency under such circumstances. Tofacilitate vibration, the separation vessel may be mounted on apiezoelectric crystal 123 connected to an electric power source forvibrating the chamber at a desired frequency.

IV. Quantitative Determinations of Biological Fluid Components

A. Rare Species Enrichment and Sample Preparation

With decreasing frequency of a target population it becomes increasinglymore difficult to reliably detect, enumerate and examine the targetpopulation. Not only is there an increasing demand on the specificity ofthe identifiers, i.e., probes or a combination of probes, but the needarises for a specific target enrichment technique in addition to theneed to process larger volumes of the bodily fluid. Table II belowillustrates this by showing the frequencies of various cell populationsamong the nucleated cells in peripheral blood of normal individuals.

                  TABLE II                                                        ______________________________________                                        Cell Frequency                                                                            Cell Number  Targets Cells                                        ______________________________________                                        1:1-1:10    10,000-1,000/μl                                                                         granulocytes,                                                                                      lymphocytes                     1:10-1:10.sup.2                                                                                  1,000-100/μl                                                                       monocytes,                                                                               eosinophils                             1:10.sup.2 -1:10.sup.3                                                                          100-10/μl                                                                                   basophils                                  1:10.sup.3 -1:10.sup.4                                                                           10-1/μl                                                                                      CD34+ cells                              1:10.sup.4 -1:10.sup.5                                                                           1,000-100/ml                                                                               CD34+, CD38- cells                            1:10.sup.5 -1:10.sup.6                                                                          100-10/ml                                                                                      tumor cells                                1:10.sup.6 -1:10.sup.7                                                                          10-1/ml                                                                                          tumor cells                              1:10.sup.7 -1:10.sup.8                                                                          1,000-100/1                                                                                  tumor cells                                  ______________________________________                                    

For analysis of infrequent cells, such as CD34+ cells or a subsetthereof, or in case of disease potential circulating tumor cells, theamount of blood needed to reliably detect, enumerate and examine thetarget population needs to be substantially larger than 1 μl. Onepractical implication for the analysis of larger blood volumes is asubstantially longer processing time. For example, for flow cytometricanalysis of a 1 ml blood sample the erythrocytes in the sample aretypically lysed, which is accompanied with a 10 fold dilution of thesample. For a typical sample flow rate of 1 μl/sec, the 10 ml volume ofthe lysed sample will thus require 2.78 hours for analysis. The need forenrichment of the target population and an increase in its concentrationis thus clearly desired. A variety of enrichment methods can be employedto increase the concentration of the desired target in the sample to beanalyzed, so that a sufficient number of target entities will be presentin the separation vessel to permit detection. Success of theseprocedures is determined by carry over of non targets, recovery oftargets, the ability to concentrate the target and the ability toaccurately analyze the target after the procedure. Introduction into aseparation vessel of a sample from a bodily fluid of which targets areconcentrated and non targets are reduced, permits enumeration andexamination of the target population.

In one method of sample preparation, an external gradient separator ofthe type described in U.S. Pat. No. 5,186,827 may be employed. Forexample, to a vessel containing 10 ml of blood, a bioactive ferrofluidis added which identifies cells of epithelial origin. After appropriateincubation, the sample is placed in the external gradient separator.After separation, the blood components not magnetically attached to thewall of the vessel are removed while the desired target substanceremains adhered to the wall of the separator. The separated cells cannow be resuspended into a smaller volume when the separation vessel isremoved from the magnetic field.

To the resuspended sample, fluorescent labeled probes can be added.After incubation, the sample is again placed in an external gradientmagnetic separator. After separation, the supernatant (including excessreagents) is removed, and the separated cells are resuspended in avolume commensurate with the chamber of a separation vessel of thepresent invention. Assuming the 10 ml of blood contained 10⁸ cells, acarryover of 0.01% would result in 10⁴ cells, which is within the rangeof the cell capture capacity of the apparatus of this invention. Given atarget cell frequency of 1 in 10⁷ and a capture efficiency of 70%, 7target cells would be captured, which is sufficient for identificationand further characterization. The cells which express the antigentargeted by the ferrofluid will align along the ferromagnetic lines inaddition to other cells which are nonspecifically bound to theferrofluid. Cells which were captured due to other reasons, such asentrapment, will not align, resulting in a further purification ofinfrequent cell types.

Identification and further characterization of the target cells can beobtained by the differences in the scattered and spectrum of thefluorescence light. An additional improvement can be achieved byutilization of a fluorescent form of the bioactive ferrofluid, thetarget cells can then be discriminated from non specifically bound cellsby the amount of fluorescence emitted by the cells, i.e. the density ofthe antigen on the cell surface of the target cell is most likelydifferent from the density of nonspecifically bound ferrofluid to nontarget cells. In contrast with flowcytometry the individual targetsisolated by means of the present invention can be reexamined, in that anoptical detection system can be configured to identify and record thelocation of the target particles of interest. Once the location of thetarget cells has been detected and recorded, the immobilization vesselthen can be examined by a more sophisticated optical detection system,such as a confocal microscope.

B. Quantitative Analysis

For conducting quantitative analysis of certain cell types, a difficultyarises from having to perform multiple dilutions of the original bloodsample prior to magnetically capturing the cell type of interest. Afterhaving performed multiple dilutions, determining the concentration ofthe captured species relative to the original blood volume requiresknowledge of the precise dilution ratio and the magnetic captureefficiency. These quantities can be determined by adding concentrationmarkers to the original blood sample.

A first marker, for determining the dilution, comprises a knownconcentration of distinctly identifiable particles that are loaded withsufficient magnetically responsive material to be captured withsubstantially total efficiency. The second marker, for determining themagnetic capture efficiency of the target cells, comprises a knownconcentration of distinctly identifiable particles that are loaded withapproximately the same quantity of magnetically-responsive material asthe target cell. The second marker can comprise magnetically responsivebeads that have been formed with sufficient magnetic material to have amagnetic moment substantially equal to that of the ferrofluid-labeledtarget entities, and similar fluid transport behavior. Alternatively,the second marker may comprise magnetically inert bodies that are coatedwith a binding substance having substantially the same number of bindingsites and binding affinity as the target cells. Other techniques can beused to provide the second marker with analogous collection behaviorrelative to the target entity. Such methods are discussed in thefollowing examples.

EXAMPLE 6

Concentration Calibrated Sample Preparation Using Magnetically-LoadedMarkers

To 10 ml of blood are added 5 ml of reagent containing an epithelialcell specific ferrofluid, 10,000 green fluorescent 10 μm beads withapproximately 500 ferrofluid particles per bead affixed thereto and10,000 red fluorescent 10 μm beads with approximately 5,000 ferrofluidparticles per bead affixed thereto. After 15 minutes of incubation, thesample is placed in a magnetic separator of the type described in U.S.Pat. No. 5,186,827 for 10 minutes. That portion of the sample notattached to the wall of the vessel is discarded, the vessel is removedfrom the magnetic field and the cells collected on the wall areresuspended in 2 ml of a solution, such as an isotonic buffer or asolution which permeabilizes the cell membrane. The resuspended cellsare placed in the magnetic field for 5 minutes and the sample notattached to the wall of the vessel is discarded. The cells collected onthe wall are resuspended in 0.2 ml of a solution containingfluorescently labeled antibodies, for example, CD45 PerCP identifyingleukocytes, anti EPCAM PE and/or anticytokeratin PE identifyingepithelial cells. Optionally, the sample may again be separated, excessantibodies discarded and the collected cells resuspended in a solutioncontaining a nucleic acid dye with fluorescence properties which can bespectrally distinguished from the fluorescence produced by thefluorescent conjugated monoclonal antibodies.

Leukocytes, epithelial cells, green and red beads can then be enumeratedby the methods described herein or by traditional enumeration methods. Ameasurement of, for example, 10 epithelial cells, 5000 green beads and7000 red beads would indicate that in case the epithelial cells have adensity of 500 ferrofluid particles/cell their concentration would be(10×10,000/5000)/10=2 epithelial cells per ml of blood; and in case theepithelial cells have a density of 5,000 ferrofluid particles/cell theirconcentration would be (10×10,000/7,000)/10=1.4 epithelial cells per mlof blood.

Although this example describes the use of two makers to accuratelydetermine epithelial cell concentration, it will be apparent that anycell concentration can be determined.

EXAMPLE 7

Sample Preparation Using Ferrofluid-Binding Markers

The cell analysis of Example 6 was repeated, except for the addition of10,000 red and 10,000 green beads with respectively 500 and 5,000antigens per bead to the blood. (Many of the antigens are cloned andrecombinant proteins can be obtained which are recognized by theantibodies). This is followed by addition of the ferrofluid whichidentifies both beads and epithelial cells. In this example, an accurateestimate of the absolute number of target cells is obtained and itdetermines in addition whether the target cell specific ferrofluidworks.

Examples 6 and 7, above, describe a sample preparation procedure tocontrol for performance of a cell analysis system, as well as indicatethe concentration of the measured target cells per volume unit. Ananalysis sample prepared according to the procedure described above canthen be quantitatively analyzed using flow cytometry, or with theapparatus described herein. The volume in the apparatus described hereinis known, whereas the volume which passes through a flow cytometer hasto be determined by the beads or by the actual measurement of the volumein which the flow cytometer measured the target events. Using precisesample dispensing techniques (pipette) the volume of the sample, thereagent and the diluent is accurately measured. In the comparableprocedure using the apparatus of the present invention, on the otherhand, accurate dispensing of sample and reagents is sufficient todetermine the cell concentration (without also requiring a count of thebeads for volume determination). In its simplest configuration, however,it is desired to obviate precise dispensing of sample. To this end, thebead approach described above can be used to determine the precisedilution of the sample, rather than the determination of the precisevolume from which the target cells are analyzed, as exemplified above.

EXAMPLE 8

Cell Concentration Determination Using Calibrated Marker Solution

Approximately 50 μl of a solution containing target cell specificferrofluid, reagents such as fluorescent nucleic acid dyes to facilitateidentification of target cells and a known concentration of 10 μm beads,for example, 1,000 per μl, with physical properties which distinguishthem from the target cells, and labeled with an amount of ferrofluidwhich is in the same range as that of the target cells, are introducedinto a separation vessel of the present invention. A drop of blood isalso introduced into the vessel by, for example, capillary action. Theblood and fluid are mixed and incubated. The vessel is now introducedinto a magnetic field and the target cells and beads are aligned alongthe ferromagnetic lines. From the number of beads counted, the actualvolume of blood which was mixed with the fluid can now be determined.For example, if 6,000 beads are measured and the chamber volume is 10μl, the volume of the marker fluid in the chamber is 6,000/1,000=6 μl.The blood volume in the chamber is thus 10 μl-6 μl=4 μl. If 32,000target cells are measured, the target cell concentration is32,000/4=8,000 cells/μl. As should be evident from the foregoingdescription, neither the exact volume of the blood nor the exact volumeof the marker fluid has to be known so long as the concentration of thebeads in the marker fluid is known and the blood and bead solution arefully mixed.

As in Example 7, beads can be added which have an amount of antigenwhich is similar to the amount of antigen on the target cells. Inaddition to the features described in examples 5, 6, and 7, thisprocedure further provides a method to determine the efficacy of thetechniques and reagents used to select and detect the target material.

C. Assessment of Red Blood Cell Parameters

Important parameters in hematology are hemoglobin and hematocrit, meancorpuscular volume (MCV), mean hemoglobin concentration (MCH), meancellular hemoglobin concentration (MCHC) and red blood cell number(RBC). To measure erythrocytes, a larger dilution of the blood isrequired as compared to leukocyte measurements or subsets thereof (onthe order of a 1,000 fold higher concentration). A higher fluid volumecan be used in the chamber in order to obtain a greater dilution. Italso could be advantageous to reduce the chamber height to reduce thenumber of erythrocytes aligned. Erythrocytes can be identified andaligned by using an erythrocyte specific ferrofluid such as glycophorinA-labeled ferrofluid or transferrin-labeled ferrofluid, the latterrecognizing only the immature reticulocytes, i.e., RNA containingerythrocytes. Erythrocytes bound to such ferrofluids can bedistinguished from nucleated cells bearing the transferrin receptor bythe absence of nuclear fluorescence. Alternatively, one could make useof the presence of hemoglobin in the erythrocytes to render theerythrocytes magnetically responsive. The iron present in hemoglobin canbe reduced, or otherwise rendered magnetic according to a knownprocedure such that the red blood cells will be immobilizable by theinternal gradients generated in the vicinity of the ferromagnetic lines.In such a method, no ferrofluid is necessary to attract the erythrocytesto the ferromagnetic lines, and the rate at which they would beattracted toward the ferromagnetic lines would be proportional to theamount of hemoglobin in the cells.

Once the erythrocytes are aligned along the ferromagnetic lines, lightscatter and absorption measurements of the individual cells can beperformed which permit the assessment of size, volume and hemoglobincontent according to procedures for conducting measurements in knownhematology analyzers, and suitably adapted for use with the presentapparatus.

The ability to assess the shape of the individual erythrocytes providesclinically useful information. Elliptocyte, leptocyte, teardrop rbc,spherocytes, sickle cells, schistocyte, acanthocyte, echinocyte,stomatocyte, xerocyte are all red blood cell shapes associated withdisease states which cannot be accurately defined by the assessment oferythrocytes in hematology analyzers. Optionally, the erythrocytes canbe further differentiated by the addition of probes which recognizeerythrocytes subsets, such as CD71 or transferrin. The fluorescence orother detectable signal of the probe could thus be analyzed through thetransparent wall member. Enhancement of the ability to assess red bloodcell shapes could be significantly improved by measurement of thesurface area of the cell membrane which can be achieved by the additionof a fluorescent membrane dye to the diluent fluid. Measurement of thetotal amount of fluorescence per cell is then proportional to the totalamount of the membrane. From the surface area of the cell membrane andthe analysis of the light scatter and fluorescence signals, the shape ofthe erythrocyte can be derived. The dilution of the blood sample can bedetermined by the addition of the beads to the diluent fluid, and thenumber and volume of the erythrocytes can be determined, the hematocrit(volume of cells/(total blood volume)) can also be determined accordingto the present method.

EXAMPLE 9

Measurement of Immune Status and Function in Whole Blood

One of the critical needs for the immune function is the ability oflymphocytes to re-circulate through the various tissues of the body.This circulation uses blood and lymph for transportation and the antigenreceptors present on the lymphocyte surface enable the monitoring of anysite where antigen could enter the bloodstream. Memory T cells specificfor antigens which are associated with specific infectious diseases suchas measles, mumps, tetanus, HIV or Lyme disease are infrequent. However,their number increases dramatically when antigen reenters the body.Monoclonal antibodies specific for disease specific T cells can be madeand upon labeling with ferrofluid the antigen (disease)-specific MemoryT cells can be aligned and enumerated. Alternately, all specific orsubsets of T cells could be aligned. The apparatus of the currentinvention could then be used to determine specific responses of cellsubsets to immunological stimuli.

Sequential flow-by reactions of generic or disease specific antigens orother markers also could be used to determine the functional status andcapability among other characteristics of the aligned cells. Aparticular advantage of the current invention is that the reaction ofindividual cells can be measured at various time points afterstimulation. This type of analysis is an obvious advantage over themeasurement of immune function by flowcytometry which permits only onopportunity to measure individual cell responses.

An arrangement for conducting "flow-by" or sequential reactions uponimmobilized target material is shown in FIG. 12. Fluid supply means,such as pumps, gravity reservoirs or syringes 220, 222 are provided forcontaining the test sample and respective reagents for reaction with thetarget material. Fluid conduits 224, 226 are provided for conductingflows of the respective fluids to a mixing valve 228. The mixing valve228 is configured to select one or more of the fluid components formixing and delivery to an inlet port 230 of the separation vessel 210.The separation vessel 210 further includes an outlet port 232 forconducting a flow of fluid out of the separation vessel 210. Theseparation vessel 210 is configured for use with magnetic arrangementsand manual or automatic optical observation means such as have beendescribed above, so that target material may be immobilized therein by aferromagnetic capture structure 218, and then subjected to sequentialreactions with the fluids provided by the fluid supply means andselected by the multi-port valve 228 in sequence and/or combination.

In a particularly preferred embodiment, the apparatus in FIG. 12 can beutilized to perform fluorescence in situ hybridization in order todifferentiate among characteristics of cell subpopulations. In such amethod, the desired types of cells are immobilized in the apparatus. Thecell membranes are permeabilized by an appropriate reagent prior to, orafter, immobilization. Then, in situ hybridization is performed on theimmobilized, permeabilized cells by conducting sequential flows ofreagents through the vessel. For fluorescent hybridization, therespective reagents comprise respective fluorescent probes.

The instant invention is particularly well adapted for performingsequential reactions due to the small size of the ferromagnetic linesrelative to other internal gradient separators. The lines can be maderelatively thin, due to the mechanical support provided by the wall ofthe vessel. Consequently, cells or other entities are rendered stronglyimmobilized by the relatively large internal gradients generated in thevicinity of the lines. Such strong immobilization force results in agreater resistance to hydraulic forces incident to conducting flow-byreactions on immobilized substances. Heretofore, magnetic capturestructures were of significantly larger dimensions in order to providean internal gradient with sufficient spatial extent to draw magneticparticles into immobilization. In accordance with the present invention,it has been found that gravitational force and/or an applied externalgradient can be employed to draw magnetizable entities toward a stronginternal gradient region generated in the vicinity of a capturestructure having relatively small dimensions, and supported along oneside of the chamber of the separation vessel.

That which is claimed is:
 1. A method of differentiating biologicalsubstances of at least a first and second type in a fluid sample,comprising the steps of:adding to the fluid sample a ferrofluid having abinding affinity for the biological substances of the first and secondtypes; placing the fluid sample in a separation vessel having atransparent wall defining one side of a chamber and having aferromagnetic capture structure supported along the transparent wall;positioning the vessel in a magnetic field for inducing an internalgradient in the vicinity of the capture structure sufficient toimmobilize the biological substances of the first and second types alongthe wall adjacent to the capture structure; immobilizing the biologicalsubstances of the first and second types along the transparent walladjacent to the capture structure; and observing the distinct opticalcharacteristics of the respective first and second types of biologicalsubstances.
 2. The method of claim 1, comprising the step of marking thefirst and second types of biological substances with respective markersto produce said distinct optical characteristics.
 3. The method of claim2 wherein said marking step comprises:adding to the fluid sample firstand second optical markers adapted for producing distinct opticalresponses from biological substances of a respective first type and asecond type; and wherein said observing step comprises the step ofilluminating the vessel with optical radiation adapted to produce saiddistinct optical responses.
 4. The method of claim 2, wherein saidimmobilizing step comprises the step of collecting the biologicalsubstances in substantially a single line along the transparent wall,and said observing step comprises observing the distinct opticalresponses through the transparent wall while the substances remainimmobilized.
 5. The method of claim 1 wherein the first and secondbiological substances are cell types of respective leukocytesubpopulations.
 6. The method of claim 5 wherein the leukocytesubpopulations comprise respective living and dead leukocytes.
 7. Themethod of claim 5, comprising the step of applying a transport force tothe vessel during said positioning step for transporting the substancestoward the internal gradient in the vicinity of the capture structure.8. The method of claim 1 wherein the first and second biologicalsubstances are respective first and second red blood cellsubpopulations.
 9. The method of claim 8, comprising the step ofapplying a transport force to the vessel during said positioning stepfor transporting the substances toward the internal gradient in thevicinity of the capture structure.
 10. The method of claim 1 wherein thefirst and second biological substances are cell types of respectiveplatelet subpopulations.
 11. The method of claim 10, comprising the stepof applying a transport force to the vessel during said positioning stepfor transporting the substances toward the internal gradient in thevicinity of the capture structure.
 12. The method of claim 1 whereinsaid observing step comprises observing distinct shapes of therespective first and second types of the biological substances.
 13. Themethod of claim 12 wherein said biological substances are cell types,and said observing step comprises enumerating the immobilized cells ofthe first and second types and determining a ratio of immobilizedsubstances relative to the net surface area of the immobilized cells.14. The method of claim 13, comprising the steps of:diluting the fluidsample with a diluent containing a fluorescent membrane dye; and whereinsaid observing step comprises measuring fluorescence from theimmobilized cells to determine the proportionate surface area of theimmobilized cells.
 15. The method of claim 1 comprising the step ofconducting sequential flows of reagents through the vessel to producethe distinct optical responses.
 16. The method of claim 15 wherein thebiological substances are respective cells of distinct cellsubpopulations, the method comprising the step of permeabilizing themembranes of the cells prior to conducting the sequential flows ofreagents through the vessel.
 17. The method of claim 16, wherein thesequential flows of reagents comprise sequential flows of reagents forproducing distinct fluorescent optical responses, and wherein saidobserving step comprises illuminating the immobilized cells withillumination selected to produce said distinct fluorescent responses.18. The method of claim 1 wherein said biological substance comprisescells and said observing step comprises conducting in situ hybridizationof the immobilized cells.
 19. The method of claim 18, comprising thestep of applying a transport force to the vessel during said positioningstep for transporting the substances toward the internal gradient in thevicinity of the capture structure.
 20. The method of claim 1, comprisingthe step of applying a transport force to the vessel during saidpositioning step for transporting the substances toward the internalgradient in the vicinity of the capture structure.
 21. The method ofclaim 20 wherein the step of applying a transport force comprises thestep of applying an external magnetic gradient to the vessel.
 22. Themethod of claim 21, wherein the immobilizing step comprises the step ofcollecting substantially a single line of the biological substance alongthe transparent wall.
 23. A method of identifying immunophenotypicsubpopulations of biological substances suspended in a fluid medium,comprising the steps of:adding to the fluid medium a quantity ofmagnetically-responsive particles having a binding affinity for thebiological substances; adding to the fluid medium a plurality of probeshaving respective distinct fluorochromes; placing the fluid medium in aseparation vessel having a transparent wall defining one side of achamber, and having a ferromagnetic capture structure supported alongthe transparent wall; positioning the vessel in a magnetic field forinducing an internal gradient in the vicinity of the capture structuresufficient to immobilize the biological substances along the transparentwall adjacent to the capture structure; illuminating the vessel withoptical radiation adapted to excite at least a selected one of thefluorochromes; and observing the optical response of the selectedfluorochrome.
 24. The method of claim 23, wherein the observing stepcomprises enumerating red blood cell subpopulations having respectivedistinct shapes.
 25. The method of claim 24, comprising the stepsof:diluting the fluid sample with a diluent containing a fluorescentmembrane dye; and wherein said observing step comprises measuringfluorescence from the immobilized cells to determine the proportionatesurface area of the immobilized cells.
 26. The method of claim 23comprising the step of applying a transport force to the vessel fortransporting the biological substances toward the internal gradient inthe vicinity of the capture structure, and said observing steepcomprises observing the immobilized substances adjacent to the capturestructure through the transparent wall.
 27. A method of differentiatingred blood cell subpopulations in a fluid sample, comprising the stepsof:rendering red blood cells in the fluid sample to be magneticallyresponsive; placing the fluid sample in a separation vessel having atransparent wall defining one side of a chamber, and having aferromagnetic capture structure supported along the transparent wall;positioning the vessel in a magnetic field for magnetizing themagnetically responsive red blood cells and for inducing an internalgradient in the vicinity of the capture structure sufficient to attractand immobilize the cells along the transparent wall adjacent to thecapture structure; and observing red blood cells immobilized adjacentthe capture structure.
 28. The method of claim 27, wherein the renderingstep comprises the step of labelling the red blood cells withmagnetizable particles.
 29. The method of claim 28 wherein themagnetizable particles are adapted to bind with a characteristicdeterminant of at least one subpopulation of red blood cells.
 30. Themethod of claim 29 wherein the magnetizable particles comprise at leastone binding substance selected from the group consisting of CD71,glycophorin A and transferrin.